The application of allostasis and allostatic load in animal species: A scoping review

Principles of allostasis and allostatic load have been widely applied in human research to assess the impacts of chronic stress on physiological dysregulation. Over the last few decades, researchers have also applied these concepts to non-human animals. However, there is a lack of uniformity in how the concept of allostasis is described and assessed in animals. The objectives of this review were to: 1) describe the extent to which the concepts of allostasis and allostatic load are applied theoretically to animals, with a focus on which taxa and species are represented; 2) identify when direct assessments of allostasis or allostatic load are made, which species and contexts are represented, what biomarkers are used, and if an allostatic load index was constructed; and 3) detect gaps in the literature and identify areas for future research. A search was conducted using CABI, PubMed, Agricola, and BIOSIS databases, in addition to a complementary hand-search of 14 peer-reviewed journals. Search results were screened, and articles that included non-human animals, as well as the terms “allostasis” or “allostatic” in the full text, were included. A total of 572 articles met the inclusion criteria (108 reviews and 464 peer-reviewed original research). Species were represented across all taxa. A subset of 63 publications made direct assessments of allostatic load. Glucocorticoids were the most commonly used biomarker, and were the only biomarker measured in 25 publications. Only six of 63 publications (9.5%) constructed an allostatic load index, which is the preferred methodology in human research. Although concepts of allostasis and allostatic load are being applied broadly across animal species, most publications use single biomarkers that are more likely indicative of short-term rather than chronic stress. Researchers are encouraged to adopt methodologies used in human research, including the construction of species-specific allostatic load indexes.


Introduction
The link between stress and health is well documented across several taxa [1][2][3]. Acute, shortterm stress is adaptive and essential for survival [3]; however, chronic or prolonged stress can have significant impacts on morbidity and mortality [4][5][6]. Most  Several of the biomarkers incorporated into ALIs in human populations are well conserved across taxa. For instance, cortisol, glucose, DHEA-S, and interleukins are frequently incorporated into ALIs in humans [5] and are also measured in animal populations [40][41][42]. Despite the overlap in the individual biomarkers that are being measured in animals and humans, the application of ALIs to non-human animals has been limited, and individual biomarkers are sometimes used as proxies instead. In the last five years, our group has called for the application of a more rigorous AL methodology in animal populations [23,43]. To better understand how the terms allostasis and AL have been used to date and where there are gaps in methodology, we conducted a scoping review of how the concepts of allostasis and AL are being applied in non-human animals.

Review protocol and expertise
This scoping review was conducted using the Arksey and O'Malley framework [44]. The PRIS-MA-ScR checklist was utilized to ensure completeness (S1 Checklist). The protocol was created in advance of the literature review based on the input and expertise of the co-authors, which include veterinary medicine (KS), biological anthropology and primatology (AE), and animal welfare (KP). The repository of relevant articles and the resulting datasets are available upon request.

Review question and scope
The overall goal of this review was to describe how principles of allostasis are being used in research with non-human animals. We had three primary objectives: 1) describe the extent to which the concepts of allostasis and AL are applied theoretically to animal populations, with a focus on which taxa and species are represented; 2) identify when direct assessments of allostasis or AL are made, which species and contexts are represented, what biomarkers are used, and if an ALI was constructed; and 3) detect gaps in the literature and identify areas for future research.

Search strategy
A comprehensive search was conducted in CABI, PubMed1, Agricola, and BIOSIS™ databases on June 5, 2021, using the following algorithm: (allostasis OR allostatic AND (animal � OR wildlife � OR mammal � OR primate � OR avian � OR bird � OR reptile � OR snake � OR lizard � OR turtle � OR tortoise � OR amphibian � OR frog � OR fish � ) NOT (human � OR hominidae OR "homo sapien")). There were no constraints on the publication date for this search.
Databases do not always search the full text, so a complementary hand-search of 14 peerreviewed journals was done. To select which journals were hand-searched, the results of the initial database search were categorized by publication and placed in rank order. Initially, the top 10 journals were going to be hand-searched. However, the 10 th ranked journal had the same number of publications as the journals ranked 11 to 14, so all 14 were included in the review. The journals that were hand-searched included: General and Comparative Endocrinology; Hormones and Behavior; Physiology & Behavior; Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology; Functional Ecology; PLOS One; Aquaculture; Integrative and Comparative Biology; Scientific Reports; Animals; Conservation Physiology; Journal of Experimental Biology; Oecologia; and Physiological and Biochemical Zoology. The website for each journal was used to conduct a full text search for the terms "allostasis" or "allostatic" and retrieve all relevant publications.

Relevance screening and inclusion criteria
The inclusion criteria for the scoping review were intentionally broad to allow for the inclusion of any relevant articles using non-human animal species. All search results were exported into Excel (Excel 2016, Microsoft Corporation, Redmond, WA). Duplicates were highlighted and removed using conditional formatting. An initial screening of the title and abstract was conducted by one author (KS). Articles written about non-human animals were included for full review, unless it was evident in the title or abstract that the animal was being used as a model for human disease. Only peer-reviewed research or review articles were included; editorials, commentaries, letters, conference proceedings, theses, and invited papers were excluded. Papers that were not available in English or could not be obtained via the Ohio State University Library system were also excluded.
After screening the title and abstract, all articles that were initially included were exported to a free citation manager (Zotero) and underwent full text review by one author (KS). Any article that pertained directly to a non-human animal population and included the terms "allostasis" or "allostatic" in the full text was included in the scoping review. Similar to the initial screening, any studies in which the animals were used as direct proxies for humans were excluded; however, laboratory animal studies were included if the research was aimed at learning about the welfare and response of the animal, and they were not used as models for humans. Articles where the search terms were listed in the literature cited or acknowledgments sections and not in other sections of the text were excluded. If the search terms were not found at all in the full text review, the publications were excluded.
To fulfill the second objective of this review, any publication that made direct assessments of AL by measuring biomarkers were coded by one author (KS). For the purpose of this review, only biomarkers that could be evaluated from ante-mortem samples (e.g., saliva, hair, feces, blood) or could be directly measured in living animals (e.g., heart rate, blood pressure) were evaluated.

Scoping review management, data charting, and analysis
All articles that met the inclusion criteria had the following data recorded: 1) taxa and species; 2) where in the article the search terms were mentioned (e.g., introduction, results); 3) whether the article was a review (including meta-analyses) or primary research; 4) which biomarkers were measured; 5) whether AL was inferred based on the biomarkers measured; and 6) if an ALI was constructed to evaluate AL.

Descriptive statistics
Following the literature search and removal of duplicates, 2,460 publications were identified for title and abstract review, out of which 1,212 articles were identified for full text review ( Fig  1). Reasons for exclusion after full text review included animals being used as models for human disease (n = 63), conceptual articles that did not specifically apply to animal populations (n = 14), the terms "allostasis" or "allostatic" being found in the bibliography only (n = 553), or the terms not being present in the full text (n = 10). A total of 572 articles met all inclusion criteria and were included in this scoping review (S1 Dataset). Of the included articles, 108 were review articles (including meta-analyses) and 464 were peer-reviewed original research publications.
Articles were written between 2003 and 2021, with 84% (479/572) published within the last 10 years (Fig 2). Since the literature search was conducted in June of 2021, the bar in Fig 2 corresponding to the number of publications in 2021 only represents half of the year. Species across all five main vertebrate groups, as well as invertebrates, were represented (Fig 3): invertebrates (n = 10), fish (n = 143), amphibians (n = 12), reptiles (n = 38), birds (n = 134), and mammals (n = 177). There were also publications that referred to animal populations, but not a specific species or taxa (n = 32) or included multiple taxa (n = 26). Most species were discussed in only one or two publications, while some species were well represented in multiple publications (S1 Appendix). The most commonly studied fish species were Atlantic salmon (Salmo salar L.) (n = 21), gilthead seabream (Sparus aurata L.) (n = 20), and rainbow trout   (Oncorhynchus mykiss) (n = 11). The most commonly studied reptile species were common lizards (Zootoca vivipara), and Eastern fence lizards (Sceloporus undulatus) with 6 publications each. The most commonly studied bird species were house sparrows (Passer domesticus) (n = 15), zebra finches (Taeniopygia guttata) (n = 7), and chickens (Gallus gallus domesticus) (n = 6). The most studied mammalian species were cows (Bos taurus) (n = 11), rats (Rattus spp.) (n = 11), and rhesus macaques (Macaca mulatta) (n = 8). An array of invertebrates and amphibian species were represented, but none that were studied in more than two publications.

Biomarkers and assessment of allostatic load
Of the 572 publications, a total of 63 (11%) measured biomarkers to make assessments of allostasis or AL, 61 primary research publications ( No Black-legged kittiwake (Rissa tridactyla) Corticosterone • The relationship between AL, which was measured using corticosterone values, and energy stores was curvilinear at both life history stages [68] No Blue fronted Amazon Parrots (Amazona aestiva)

Glucocorticoids
• Dominant birds had higher AL than subordinates, indicated by higher fecal glucocorticoid metabolites [69] No

European starlings (Sturnus vulgaris)
Corticosterone HR • Decreased temperatures and exposure to rain caused elevated heart rates and corticosterone levels in non-molting starling indicating that these birds had increased AL [70] No Griffon vulture (Gyps fulvus) Cortisol DHEA • Cortisol and DHEA levels were higher in the feathers of physiologically compromised birds compared to the healthy control group • Moulted feathers can be a non-invasive means of evaluating AL in birds [71] No Mallard ducks (Anas platyrhynchos) Corticosterone • Feather corticosterone reflected energy expenditure and was therefore a proxy of AL • Feather corticosterone provided retrospective information about AL early in life that was not detected from the body condition of birds in real time [72] No Red crossbill (Loxia curvirostra) Corticosterone Corticosterone binding globulin capacity Testosterone • No association was seen between parasite load and corticosterone levels • Conclude that parasite infections did not affect AL in this species [73] No

Red kites (Milvus milvus)
Corticosterone DHEA • Free-living kites had higher levels of corticosterone than captive birds, indicating a higher AL [74] No

Mammals
Assamese macaques (Macaca assamensis) Glucocorticoids • During the mating season females that were in closer association with males had lower fecal glucocorticoids • In the non-mating season positive female-female socializing resulted in lower fecal glucocorticoids • Positive social interactions resulted in lower AL based on glucocorticoid measures [75] No Barrow Island euro (Macropus robustus isabellinus) Cortisol Hb Leukocyte count LVP Osmolality PCV RBC Reticulocyte count • When exposed to prolonged drought animals experienced AL, but were able to maintain their normal homeostatic state • Conclusions were based on elevated osmolality, cortisol and LVP [76] No Black capuchins (Sapajus nigritus) Glucocorticoids • Decrease fruit intake during the dry season resulted in increased fecal glucocorticoids in juvenile males • Glucocorticoid levels in adult males was more impacted by breeding season • Fruit consumption as the main source of AL for immature animals, while reproductive costs had greater effect on adults [77] No Blue monkey (Cercopithecus mitis) Glucocorticoids • Directly equated AL with deviation of fecal glucocorticoids from the baseline • Participating in social play may decrease social uncertainty and AL [78] No Brush-tailed bettong (Bettongia penicillata) Glucocorticoids • Fecal glucocorticoids were not elevated after wildfires indicating that bettong maintain allostasis in the period immediately following a fire [79] No Cairo spiny mouse (Acomys cahirinus) Glucocorticoids • There is a significant interaction between parasitism and social contact • Solitary females with no parasitism had the highest glucocorticoid levels • Social contact and parasite infestation may lessen AL in pregnant rodents [80] No Capuchin monkeys (Sapajus libidinosus) Cortisol Testosterone • Dominant males had higher basal and peak fecal cortisol levels, and therefore had higher AL than subordinates [81] No Cheetah (Acinonyx jubatus)

Glucocorticoids
• No difference in fecal glucocorticoid levels between parous and nulliparous females • AL may not impact reproductive success [82] No Cortisol • Cheetahs had lower serum cortisol concentrations than leopards • Capture may cause a higher AL in leopards [83] No Chimpanzees (Pan troglodytes) Cortisol • Anesthesia for routine examinations resulted in increased urinary cortisol • Findings indicate a major disruption of homeostasis and an AL [84] No Dog (Canis familiaris) Cortisol DHEA-S • Maternity plays a role in HPA axis activation resulting in chronic cortisol secretion leading to increased AL [85] No (Continued ) • Sheep that were chronically stressed by individual housing and sleep deprivation showed HPA-axis dysregulation, suggesting an increased AL in the chronically stressed group of animals [98] No Spotted hyena (Crocuta crocuta)

Glucocorticoids
• Variations in fecal glucocorticoids based on dominance provide evidence that hunger and sibling competition affect AL in spotted hyenas [99] No Glucocorticoids • During breeding periods when there was interaction between male competitors, the low-ranking males had higher fecal glucocorticoids than high-ranking males • There was no difference in fecal glucocorticoids between the groups when individuals were alone or competitors were absent • Differences in AL across male social ranks may be a result of interactions with other males [100] No White faced capuchins (Cebus capucinus) Dihydro-testosterone Glucocorticoids Testosterone • Compared to subordinate males the dominant males have higher fecal testosterone, dihydrotestosterone and fecal glucocorticoids • Regardless of dominance status all males had elevated fecal glucocorticoids in the presence of fertile females • Findings suggest that there is a cost of dominance, but that in the presence of fertile females AL increases for all males [101] No Yellow-bellied marmots (Marmota flaviventris)

Glucocorticoids
• Young marmots had lower fecal glucocorticoids in rural environments, therefore lower AL • Adult marmots had higher fecal glucocorticoids in rural environments • All age groups exhibited parabolic relationships between degree of urbanization and fecal glucocorticoids [102] No A total of 61 primary research publications that made direct assessments about allostasis and/or allostatic load in non-human animals, including animal class, species, biomarkers evaluated, major conclusions and whether an allostatic load index (ALI) was constructed.

Discussion
The overall goal of this scoping review was to evaluate the extent to which principles of allostasis and AL are being applied to non-human animals. Of the 572 articles included in this review, most were written within the last ten years. Over the last 5 years, there have been 40-60 peerreviewed publications annually. This change reflects a growing application of allostasis and AL in animal populations.
The species diversity encompassed all taxonomic groups, with over 250 different species represented across the 572 articles. Seventy-nine percent of the publications discussed mammalian, fish, or avian species, with far fewer papers focusing on reptiles, amphibians or invertebrates. This gap in the literature presents opportunities for future research, particularly in taxa like amphibians, which are facing global population declines due to diseases like Batrachochytrium dendrobatidis (Bd) [103]. Since many disease states are diagnosed and/or monitored by measuring biomarkers (e.g., cholesterol and insulin resistance for metabolic syndrome in humans), ALIs encompassing multiple somatic systems have the potential to predict future health outcomes. The link between stress and risk of disease is well described in a wide variety of species, and ALIs have been used to characterize disease risks in human populations [104,105]. Additionally, ALIs have the potential to be used to assess the impact of social and environmental stressors and how they drive animal movements and affect disease ecology on a broader scale [106]. Even within the largely represented groups, like fish, there is potential for expanded applications of AL. Many of the fish publications focused on the health and welfare of commercial species in aquaculture settings [54, 58, 107], with 61 publications looking at Atlantic salmon, gilthead seabream, rainbow trout or European seabass (Dicentrarchus labax). In contrast, only three papers explored allostasis in zebra fish (Danio rerio) [108][109][110], which are an important laboratory species that may benefit from studies of AL.
Despite 572 publications mentioning allostasis or AL, only a small proportion (63 publications, 11%) made direct assessments using physiological biomarkers. The other 509 articles only mentioned AL hypothetically as part of the introduction or discussion of other findings. Of this subset of 63 articles, 61 were primary research studies and two were meta-analyses that incorporated data from multiple publications to make their assessment. Within the primary research, 49 different species were studied across all taxa, with the exception of amphibians.
There were common themes amongst the 63 publications, including a focus on environmental challenges, social structure, and animals under managed care. Evaluating AL in the context of environmental challenges was a focus of several publications and encompassed different focus areas including the impacts of human activities (i.e., agriculture [51], urbanization [102], snow sports [14], and pollution [74]), and characterization of the effects of environmental parameters on AL (i.e., weather [70], fire [79], and salinity [47]). Several publications investigated the effects of social structure on AL in several species including hyena [100], Assamese macaques [75], bearded capuchins [81], cichlids [60], and rainbow trout [52]. Multiple researchers aimed to investigate the connection between animal management techniques and AL [53,95], in some cases with an explicit emphasis on animal welfare [54,92].
While each of the 63 publications drew direct conclusions about AL from their findings, the data need to be interpreted with caution. One of the biggest methodological challenges was that a high proportion (44%) of studies made conclusions about allostasis or AL based on glucocorticoids alone. This finding is unsurprising, as glucocorticoids have historically been considered the principle hormonal mediator for AL [26,111,112]. However, there are limitations to using glucocorticoids as the only measure of stress [113,114]. For example, substantial inter-and intra-individual variation, as well as fluctuations due to temporary coping mechanisms associated with season or reproductive effort, complicate their interpretation [115][116][117][118]. Moreover, research in humans indicates that individual biomarkers are inadequate for estimating AL [119][120][121][122][123]. Therefore, given the advances in methodology, conclusions about AL cannot be made based on glucocorticoids alone.
A second consideration in interpreting the findings of many of these 63 publications is the use of AL to evaluate response to an acute stressor, often using a single-biomarker like glucocorticoids (e.g., [53,70,71,85]). This deviates from the original purpose for the development of ALIs in humans, which was to estimate wear-and-tear as the result of chronic, long-term stress. Even when estimated using multiple biomarkers, AL should not be used to replace glucocorticoids as a measure of an acute stress response. For instance, Arlettaz et al. (2015) characterized the impact of free-riding snow sports had on black grouse (Tetrao tetrix) in an alpine habitat. To mimic the disturbance of sports, grouse were flushed on consecutive days and subsequently had elevated fecal glucocorticoids compared to baseline. Based on these findings it was concluded that repeated disturbances resulted in an increased AL and thereby presented a threat to wildlife populations. Similarly, Hing et al. (2016) investigated the effects of wildfires on brush-tailed bettongs (Bettongia penicillata) by measuring fecal glucocorticoids two days after a fire. When there was no significant elevation of glucocorticoids compared to baseline, it was concluded that this species adapts to these environmental challenges with no effect on AL.
While these types of evaluations are essential to monitor high-risk populations, neither study adequately assessed AL, as the conclusions were based only on changes in cortisol levels occurring over a short time period.
Although ALIs have traditionally been used to evaluate the impact of chronic, long-term stressors in humans, there are challenges with this approach in non-human animal species, particularly wildlife, as it can be difficult to disentangle acute stress from chronic stress. For example, animals must be manually or chemically restrained to obtain a blood sample, which likely increases some of the biomarkers of interest, such as cortisol and glucose [124,125]. Additionally, repeated sampling of animals can be challenging in wild settings, making it difficult to identify all the potential stressors an animal encounters over time. While it would not be an estimate of AL researchers may be able to adapt the methodology to try and consider using an index of biomarkers that would be expected to increase in the face of acute, shortterm stress, such as glucose, cortisol, and catecholamines. This approach can allow us to gain a more robust understanding of the "cost" associated with acute stress in animals compared with single biomarkers such as cortisol [126].
Only six of the 63 publications that made direct assessments about AL used an ALI following the original method used in human research [31]. Western lowland gorillas (Gorilla gorilla gorilla) were the only non-human primate species in which an ALI was constructed and used to evaluate AL in four articles by the same research group [86][87][88][89]. Seven biomarkers were measured for the gorilla ALI, including albumin, cortisol, corticotropin releasing hormone (CRH), DHEA-S, glucose, IL-6, and tumor-necrosis factor (TNF)-α. Older animals, males, and gorillas with a higher number of stressful events over their lifetime had higher AL [86]. In a follow-up study, the authors found that wild-caught female gorillas had higher AL than mother-reared gorillas, although there was no difference in AL by rearing history for males [87]. Building upon their initial model, the authors found that the associations of AL with sex, age, stressful events, and rearing history remained when additional institutions were incorporated [88]. The authors later expanded the ALI and found that adding cholesterol and triglycerides improved predictions of morbidity and mortality risk in zoo housed gorillas [89].
In another publication that constructed an ALI, the authors proposed a measure of AL that they referred to as the "rat cumulative allostatic load measure (rCALM)" [95]. This study included the following biomarkers: cortisol, blood glucose, body weight, interleukin-1β (IL-1β), interleukin-2 (IL-2), IL-6, leptin, lactate, and creatine. The authors found that when evaluated individually the biomarkers were not predictive for neuronal deficits. However, when used as a comprehensive ALI, rCALM was an effective predictor of neurologic deficits. The authors concluded that the rCALM index estimated the effects of chronic stress and could potentially be used to indicate future disease risks.
The last publication that constructed an ALI used a meta-analysis. The aim of this publication was to evaluate hypotheses that explain variation in parasitism based on social status in vertebrate species [45]. The authors combined data from multiple studies and determined AL based on previously described methodology [46]. Authors concluded that AL was not correlated with relative parasitism in vertebrates [45]. However, the Goymann and Wingfield (2004) method used for calculating AL in this study deviates from the standards used in human populations. Instead of determining AL using an ALI comprised of multiple biomarkers, AL scores were assigned to individuals based on the assumed costs of becoming dominant within a social group; assessments were then made based on each individual's assigned AL [46]. These assumed costs of dominance were based on cortisol levels in dominant vs. subordinate animals. However, cortisol alone is an insufficient proxy for AL, making this methodology problematic. Future meta-analyses that incorporate biomarkers measured in a species across multiple publications are encouraged.
Many of the papers reviewed here, both those that made direct assessments about AL and those that did not, measured multiple biomarkers, but assessed them individually and not as an index (e.g., [41,49,127]). Thus, there is an opportunity to re-assess previously collected data as an ALI, which may increase the power and impact of the data. For instance, Hudson et al (2020) measured six different biomarkers in Colorado checkered whiptail (Aspidoscelis neotesselata) that reflected reproductive status, energy metabolism and innate immunity. The authors assessed biomarkers individually to determine if there were AL changes based on season and reproductive status [62]. Instead, authors could have used these biomarkers to create an ALI to assess AL.
Since there is biological variation between taxa, there is likely not one single set of biomarkers that universally applies to all species, although it may be possible to determine a single ALI for a group of closely related species (e.g., great apes). Instead, we recommend that species-specific ALIs be constructed using biomarkers that are most reflective of long-term stressors. Biomarker discovery and advances in animal endocrinology are required to identify sufficient biomarkers to construct ALIs in many species. For example, we recently published a study that constructed an ALI to measure AL in ring-tailed lemurs (Lemur catta), and one of the largest challenges was finding biomarkers that could be measured in lemur serum with valid results [128]. In our case, several inflammatory cytokines were investigated as potential biomarkers for incorporation in the ALI but could not be reliably measured using the commercially available assays. It is important to note that one of the limitations of incorporating new biomarkers in an ALI is the lack of information about normal reference ranges in many species. Even when reference ranges are available, measured concentrations may be affected by the chemical restraint necessary for sample collection. However, researchers often uses sample-based cutpoints instead of normal reference ranges to calculate AL; thus, an absence of reference ranges does not necessarily preclude the use of a biomarker for this type of research.
Future research using non-human animals should adopt a new way of thinking when assessing allostasis and AL. First, we encourage researchers to refrain from making conclusions about allostasis using glucocorticoids alone, and to focus on chronic rather than acute stressors. Second, we suggest that an ALI be constructed for each species using multiple relevant biomarkers that ideally reflect neuroendocrine, cardiovascular, immune, and metabolic systems as originally proposed for humans by Seeman et al. (1997). Using this approach, AL may become a useful measurement of stress and animal welfare. Indeed, several authors reflected on the importance of continuing to develop these measures as means of potentially evaluating chronic stress in animal populations [29,82,129] and acknowledged that AL may be an important tool in assessing animal welfare [43,130]. The next step is to continue to refine approaches and methodologies to create practical and appropriate ALIs across species.
There are some limitations to the generalizability of this review. We only included manuscripts published in English, which excluded potentially relevant literature published in other languages. We also excluded studies that used laboratory rodents as models for humans, which may have limited the number of studies using common laboratory animals such as rodents and primates. Finally, "invertebrates" was not used as a specific search term; however, several invertebrate publications were found in the search results and included in the review. As a result, this review likely underestimated the number of papers that are applying allostasis or allostatic load to invertebrate species.

Conclusion
This review describes how principles of allostasis and allostatic load are being used in research with non-human animals. We identified a total of 572 peer-reviewed publications that mentioned allostasis and/or allostatic load published since 2003, covering a variety of nonhuman animal species. Of these, 63 made direct assessments about allostatic load in animals. However, many of these assessments were based on single biomarkers, such as glucocorticoids, and were focused on the effects of acute rather than chronic stressors. Future research in animals is encouraged in this area, with an emphasis on the creation of allostatic load indexes. Researchers should also use more consistent methodologies when assessing allostatic load, such as those already established in human research.